PROJECT SUMMARY Major depressive disorder (MDD) is a disabling, often chronic, mental illness with not fully understood pathophysiology and high treatment resistance to currently available antidepressants. Accumulating evidence suggests that glial and glutamate synaptic abnormalities play an essential role in the pathophysiology of MDD and may offer a powerful treatment target. However, a major obstacle in the field is the paucity of human in vivo biomarkers of glial function and glutamate neurotransmission; markers that could serve as indices of target engagement and target validation. These biomarkers are urgently needed to facilitate early stages of drug development, and perhaps reduce the failure rate of clinical trials. The proposed project capitalizes on recent advances in 13C magnetic resonance spectroscopy (13C MRS) to examine in vivo glial and synaptic functions in MDD, and to establish the potential utility of 3 biomarkers: 1) glial oxidative metabolism (VTCAg), a translational cell specific biomarker that was previously related in rodents to depression pathology and treatment, but never tested in humans. 2) Neuronal energy production over glutamate cycling (VTCAn/Vcycle), a marker of glutamate neurotransmission highly conserved across animals and humans that is resilient to various potential neuroimaging confounds. 3) Global brain connectivity (GBC), a novel graph-based resting state functional connectivity MRI (rs-fcMRI) measure that was recently shown to be altered in MDD and to be highly sensitive to treatment response to the glutamate modulator ketamine, a rapid acting antidepressant. The proposed project will complete rs-fcMRI and prefrontal 13C-acetate MRS scans in 3 study groups: 1) MDD patients on stable treatment with a serotonin reuptake inhibitor, who are resistant to their treatment regimen and continue to suffer from significant depression symptoms (SRI), 2) un-medicated MDD patients currently depressed (nSRI), and 3) healthy controls free of psychopathology (HC). These studies will aim to demonstrate reduced prefrontal VTCAg and VTCAn/Vcycle in depressed patients regardless of treatment status. This will be interpreted as evidence of altered prefrontal glial metabolism and synaptic transmission. It will also suggest that these biomarkers may be sensitive to treatment failure. The project will also aim to demonstrate altered prefrontal global connectivity regardless of treatment status, while most importantly providing new evidence of a link between synaptic measures of glutamate neurotransmission and large scale intrinsic networks. Together, the data will apply novel tools in the study of MDD and, if the study hypotheses were confirmed, the provided evidence could offer unique biomarkers of glial and synaptic function in MDD. Furthermore, considering the critical role of glial and glutamate neurotransmission in brain functioning, the specific data generated in MDD could be of relevance to several psychiatric illnesses, in particular stress-related disorders.